The present invention relates to a method and apparatus for simultaneous quantification of the amounts of one or more radioactive nuclides within arbitrary regions on a surface where these nuclides have been deposited, adsorbed or fixed. These radioactive nuclides serve as markers on compounds that typically have been incorporated into tissue sections or into larger biological molecules that by various mechanisms have been bound to chemical substances on this surface. The method is especially well suited for DNA microarray deductions through the use of nucleotides labelled with different beta-emitting radionuclides.
It is widely believed that thousands of genes and their products, i.e. RNA and proteins, in a living organism function in a complicated and orchestrated way that creates the mystery of life. However, traditional methods in molecular biology generally work on a xe2x80x9cone gene in one experimentxe2x80x9d basis, which makes it hard to achieve the overall picture of the gene function. Thus, biological microarrays represents one of the most potent new tools in biological research to emerge in recent years, since it gives the opportunity to study a complete set of genes and their products simultaneously.
A microarray is an ordered arrangement of biological molecules immobilised in sample spots on a test plate which provides a medium for matching known and unknown samples of biological molecules. The immobilised molecules on the test plate are often denoted probe molecules, while the biological molecules from the test samples are denoted target molecules. In the case when the probe molecules and target molecules forms specific complementary pairs of biological molecules, the ordered arrangement of the test spots can be employed to identify specific biological molecules in a test sample from an organism and also to determine the abundance of these molecules. Typical examples of complementary biological molecules are hybridisation pairs of DNA, gene-anti gene etc. In microarrays the sample spots are typically less than 200 xcexcm in diameter, but there xe2x80x9cmacroarraysxe2x80x9d with sample spots with diameters of typically 300 xcexcm or larger have been described.
The biological microarray technique can be applied for numerous applications such as diagnosis, identification/discovery of new genes and proteins, drug discovery, pharmacological and toxicological research etc. The technique can also be employed to comparison tests where biological components from several sources are adsorbed onto the same array, for instance from a healthy cell and a tumour cell.
Among biological microarrays, it is especially one type that has drawn attention the latest years; the DNA-microarray. This technology promises to monitor the whole genome on a single chip, and thereby make it possible to acquire a better picture of the interactions between thousands of genes simultaneously.
In general, the conventional method of determining biological activity by DNA-microarrays can be described as follows; strands of probe cDNA (typically 500-5000 bases long) are immobilised in a specific and ordered array onto a solid plate (typically a glass plate). Then the probe cDNA is exposed to one or several targets (marked cDNA from the test samples) either separately or in a mixture. The targets, labelled cDNA, are produced enzymatically by reverse transcriptase from samples of RNA which are extracted from the test samples and labelled with specific marker molecules. The reverse-transcribed RNA transcripts of the samples are hybridised with the probe cDNA on the microarray. Thus the amount and type of each target cDNA can be determined by measuring the location and concentration of each marker molecule at each test spot on the microarray, since the marker signal from each test spot reflects the relative transcript amounts for each specific transcript at each test spot on the microarray. To eliminate sample variation, the signal ratio between two competing samples is the preferred measurement.
Fluorescence Tagged Nucleotides
Traditionally, the detection of signals using this technology has been based on in vitro incorporated nucleotides labelled with suitable fluorophores, that is, specific fluorophores (fluorescence molecules) of a distinctive colour are inserted into the RNA extracted from each sample, respectively. Thus nucleotides labelled with fluorophores of a distinctive colour are incorporated into the target cDNA which will be hybridised to the probe cDNA. The fluorophores may be excited by different wavelengths, and similarly also emit at different wavelengths. Laser light and the use of appropriate filters to separate signals from two or more cDNA populations will generally achieve this.
However, the general need for starting material is in the range of 50 xcexcg of total RNA, or approximately 5xc3x97107 cell equivalents. This relatively high amount of material excludes the use of standard technology from a number of very relevant applications, including clinical diagnostics. One highly significant factor in this lack of sensitivity is the low incorporation rate of current fluorophore-tagged nucleotides in the reverse transcription of cDNA from RNA. Thus only a relatively low number of fluorescence molecules will be incorporated per synthesised cDNA. Also, applications requiring incorporation of fluorescence through cell culture will be excluded, as fluorophore-tagged nucleotides will generally not be included through the cellular machinery. Emerging techniques to achieve better signal strength include enzymatic amplification of sample material and chemical signal amplification. Such methods demonstrate that it is possible to reduce sample size.
Amplification techniques have recently been published allowing a reduction in sample size down to 100 ng total RNA starting material (Wang, E. et al. Marincola FM Nat Biotechnol 2000.18(4):457-459). This is achieved through one or more rounds of cycling between RNA and cDNA. In this reaction it is possible in each round to obtain approximately a 50-fold increase of the material by attaching a T7 promoter at one end to enable generation of RNA, and at the other end exploiting a feature of certain reverse transcriptases to add a specific primer to all cDNAs at most 5xe2x80x2 end at the time of first reverse transcription. This feature is necessary to avoid generation of shorter length cDNAs.
An alternative strategy is to amplify the signal from the test material through chemical means. The most sensitive strategy so far available relies on nested tangles of labelled branched synthetic DNA molecules (Nilsen, T. W. et al. J. Theor. Biol. 1997.187:273-284; Wang, J. et al. Electroanalysis 1998.10:553-556; Wang, J. et al. J. Am. Chem. Soc. 1998.120:8281-8282). These may be bound to poly-A tails of cDNA prior to array hybridisation. Generally, a 250-fold increase in signal strength may be achieved.
Neither of these strategies have been rigorously tested for reliable performance and sensitivity levels. It is likely that amplification techniques will lead to degrees of bias of the starting material, due to the enzymatic nature of the process combined with the large variation in mRNA length for different transcripts.
The large amount of test material necessary to achieve adequate signal strength, and the problem that the available fluorophores are accepted only with difficulty by the reverse transcriptase enzyme represents thus considerable disadvantages in the prior art.
Radioactivity Labelled Nucleotides
It is known that the problem with low acceptance by the reverse transcriptase enzyme can be solved by employing radioactive isotopes for the labelling of nucleotides.
Historically, radioactive isotopes have been in widespread use for sensitive detection and quantification of nucleic acids. The common use has been confined to the use of one single, usually beta-emitting, radionuclide, incorporated either into a probe (detector nucleic acid) or directly in vivo. The detection has been performed using liquid scintillation or gamma counters, and in the case of a two-dimensional distribution (e.g. Northern and Southern blots), autoradiography film, phosphor imagers, and digital autoradiography systems has been employed (ref. French patent BioSpace, Cern patent, Charpak).
Measurements of the distribution of a radioactive compound in a thin section of tissue or Southern and Northern nucleic acid blots has traditionally been performed with film autoradiography, and this method still remains the common choice for fine resolution studies. However, it suffers from a limited dynamic range (only 1.5-3 orders of magnitude) and low sensitivity, is laborious and gives inaccurate determinations.
Storage phosphor screen imaging systems have a much better sensitivity than film autoradiography, 250 times more sensitive than X-ray film with 32P and 60-100 times more sensitive than direct film autoradiography with 14C and 35S. The linear dynamic range of these systems are 4-5 orders of magnitude and quantification methods give results that are much more reliable than with film. However, areas having activities below the minimum threshold are not quantified accurately, and inevitably imposes a limitation to the accuracy in measurements of activity distributions. A major practical problem of the two latter radiography modalities is the need to know the exposure time before acquiring the picture so that underexposure or overexposure may be avoided. Especially with film, where exposure times may be months, a bad estimate of the exposure time may cause waste of a considerable time.
The problems associated with the two latter radiography modalities can largely be solved by employing digital systems with direct event detection. This give results with absolute linearity, the count rate is only limited by the speed of the electronics, and the need for accurate estimates of exposure times is eliminated as one can inspect the cumulative pictures at any time. Even though the geometrical resolution of the available systems cannot compete with that of the film technique, it is comparable with phosphor screen imaging systems.
However, these techniques can not separate the contributions from different non-monoenergetic nuclides, and thus cannot be applied for simultaneous quantification of two or more different radioactive emitters at the same spot on a biological microarray. Obviously, this impedes (hinders) every application where one wants to simultaneously compare the activity from more than one specimen, such as for instance a healthy cell and a tumour cell. Thus a whole class of important diagnostic and research applications is cut off from the benefits of tagging nucleotides and/or other biological molecules with radioactive isotopes.
Also, according to our knowledge, the known methods and apparatuses for performing the conventional radiography methods are all limited to labelling all molecules on the surface with only one radio nuclide at time and a limited number of radio isotopes. Thus there is a need for a general method for performing simultaneous determinations of the radioactivity/intensity of two or more different radio nuclides at each spot in a biological microarray. A gamma camera equipped with a parallel hole collimator may be used to simultaneously measure the distribution of several gamma-emitting radio nuclides on a surface. However, this camera cannot resolve structures with spatial extent on the sub millimeter scale.
The main objective of this invention is to provide a method and apparatus for performing simultaneous quantifications and/or comparisons of one or more target biological molecules that are distinctively marked with radioactive isotopes and then adsorbed onto a probe array of biological molecules deposited on a substrate, and which substantially eliminates or reduces the above mentioned problems.
An additional objective of this invention is to provide a specific method and apparatus for simultaneous determination of two or more cDNAs which are labelled with beta emitting radionuclides with distinctive distributions, and which then are adsorbed onto a DNA-microarray.